Training of Neutrophils to Defend Against a Lethal Bacterial Infection of the Lung

/ABSTRACT Hospital-acquired infections are an increasing problem, especially in intensive care units (ICUs), for which the Gram-negative bacterium, Pseudomonas aeruginosa, poses a serious threat. In patients on life support, P. aeruginosa can cause ventilator-associated pneumonia (VAP) that is associated with significant mortality Despite access to new antibiotics, multidrug resistance to P. aeruginosa has doubled over the past 30 years precipitating an urgent need for alternative strategies for host defense against this pathogen. We undertook a study with a goal to establish a strategy to improve host defense against PA. We reported that a single low dose of ultrapure LPS does not induce tolerance but instead the low dose LPS trains the immune system to mount an effective defense against a lethal P. aeruginosa infection (strain used PA14) with 100% survival. Without prior LPS exposure, PA14 infection did promote a significant influx of neutrophils to the lung, and yet the mice succumbed to infection. 2-3 log higher bronchoalveolar lavage (BAL) cells, largely comprising neutrophils, were recovered from LPS+PA14 mice compared to that from PA14 mice. This suggested that low level LPS pre-treatment trains lung neutrophils to migrate to the alveolar lumen to effectively handle the infection. Notably, both mouse and human studies highlight the importance of P. aeruginosa clearance from the airspaces for increased survival. Investigations of chemokine receptors that might account for the significantly higher numbers of BAL neutrophils in LPS+PA14 mice show differential expression of CXCR4 in BAL neutrophils of LPS+PA14 but not PA14 mice. scRNA-seq analysis of lung innate immune cells unraveled a novel finding that LPS promotes the expansion of a neutrophil population, N3, uniquely enriched in interferon (IFN)-stimulated genes (ISGs), phagocytosis-associated genes and a cell-killing gene set comprising multiple genes with anti-bacterial properties including Lgals3, which encodes galectin-3 (gal-3). Analysis of LPS+PA14 gal-3 knockout (KO) mice revealed a significantly higher bacterial burden in the BAL neutrophils compared to that in control WT LPS+PA14 mice. Also, our ongoing studies show that the LPS+PA14 gal-3 KO mice succumb to infection after 24h. Cellular metabolism being integral to immune training, examination of gene expression related to cellular metabolism has revealed selective expression of the glutamine (Gln) transporter, Slc38a1 in N3 neutrophils and functional analysis supports Gln metabolism in lung neutrophils of LPS-trained mice. Taken together, these observations lead us to hypothesize that LPS-induced training to defend against a lethal P. aeruginosa infection involves expansion and sustenance of a neutrophil population, N3, that migrates to the airspaces in a CXCR4-dependent fashion and engages gal-3 and Gln metabolism to rapidly clear bacteria and promote host survival. We propose the following two aims to address this hypothesis: Aim 1. To determine the role of CXCR4 and gal-3 in LPS-induced protective function of airspace neutrophils. Aim 2. To determine whether LPS-induced protection and training of lung neutrophils involves Gln metabolism. Project Number: 1R21AI193306-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Prabir Ray | Institution: UNIVERSITY OF PITTSBURGH AT PITTSBURGH, PITTSBURGH, PA | Award Amount: $430,760 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 IIDA-B (90)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19330601